As sandwich cultured (SC) hepatocytes can repolarize to form bile canalicular networks, allowing active excretion of compounds in a vectorial manner, the model has been widely used for assessing the transporter related complexity of ADME/tox issues. A lack of quantitative information on transporter expression during cell culture has made in vitro to in vivo extrapolation of hepatobiliary transport difficult. In the present study, using our newly developed LC-MS/MS absolute quantitative methods, we determined the quantitative expression profile of three biliary transporters in SC rat and human hepatocytes. A significant shift of hepatobiliary transporter proteins was observed both in human and rat sandwich cultures. A decrease of BSEP/Bsep protein and an increase of BCRP/Bcrp protein were detected in both rat and human hepatocytes over time in culture. Interestingly, Mrp2 in rat hepatocytes was significantly diminished, while MRP2 constantly increased in human hepatocytes during the cell culture. Consequently, the interspecies difference between rat and human in absolute amount of MRP2/Mrp2 was minimized over time in culture. Following the sandwich culture, the species difference of hepatobiliary transporter protein between human and rat at day 5 post SC was diminished (MRP2/Mrp2), identical (BSEP/Bsep) or reversed (BCRP/Bcrp), compared to the in vivo situation. In addition, the absolute protein amount of BCRP/Bcrp or MRP2/Mrp2 was proportionally correlated with the intrinsic biliary clearance estimated in various lots of SC rat and human hepatocytes. The results revealed that absolute protein amount is a key determinant for hepatobiliary clearance and could provide fundamental support on extrapolation of biliary secretion from in vitro to in vivo.